Many different techniques have been developed in the field of fluorescence microscopy to restrict excitation light to a thin region of a specimen near the coverslip in order to improve the signal-to-background noise ratio and the spatial resolution of the specimen features or components of interest. Conventional widefield and laser scanning confocal fluorescence microscopy are widely employed techniques that rely on illumination of fluorophore-labeled specimens with a broad cone of light. The limited spatial resolution demonstrated by widefield fluorescence microscopy, especially along the optical axis, often renders it difficult to differentiate between individual specimen details that are overpowered by background fluorescence from outside the focal plane.
In contrast, total internal reflection fluorescence (TIRF) microscopy employs the unique properties of an induced evanescent wave to selectively illuminate and excite fluorophores in a restricted specimen region immediately adjacent to a glass-water (or glass-buffer) interface between the specimen and a transparent substrate.
The basic concept of total internal reflection fluorescence (TIRF) microscopy is simple, requiring only an excitation light beam traveling at a high incident angle through the solid glass coverslip or dish, where the cells adhere.
FIG. 1 illustrates an example of the basic concept of TIRF microscopy. Illumination light 100 is transmitted into a substrate 102, such as a coverslip, a coverplate or a slide. The illumination light 100 strikes an interface 104 between the substrate 102 and a specimen or sample 106 to be imaged at a nonzero angle of incidence 108 with respect to the interface normal. When the refractive index n2 of the specimen 106 is lower than the refractive index n1 of the substrate 102, that is n2<n1, and when the angle of incidence 108 is greater than or equal to the critical angle of the interface 104, with respect to the interface normal, the light experiences total internal reflection. Thus, none of the illumination light 100 can pass into the specimen 106 and all of the illumination light 100 is reflected back into the substrate 102. However, the reflected light generates an evanescent wave with the same wavelength as the illumination light 100. The electromagnetic field of the evanescent wave penetrates beyond the interface 104 into the specimen 106 and excites fluorescence within a thin region of the specimen 106 near the interface 104. The intensity I of the evanescent field decays exponentially with increasing perpendicular distance z from the interface 104, as illustrated in FIG. 1 and as described by equation 1:I(z)=I(0)e−z/d  (1)
where I(z) represents the intensity at a perpendicular distance z from the interface 104, where I(0) represents the intensity at the interface 104, and where d represents the characteristic penetration depth at a wavelength λ of incident light in a vacuum. The characteristic penetration depth d is expressed by equation 2:d=λ/(4π·sqrt(n12 sin2 θ1−n22))  (2)
Typical penetration depths are only about 100 nanometers from the interface 104, as represented by the dashed line 112 in FIG. 1. Fluorophores of fluorescently labeled components located within the vicinity of the interface 104 can be excited by the evanescent field. A portion of the fluorescent light emitted from fluorophores near the interface 104 may be captured by an objective lens and may be used for fluorescent imaging of the specimen 106. Accordingly, this technique is useful for studying phenomena near the interface 104 between the substrate 102 and the sample 106, since other parts of the sample 106 are not illuminated at all.
FIG. 1 illustrates a schematic representation of an objective 114 used to illuminate the specimen 106 disposed on the substrate 102. The objective 114 is an oil immersion objective with immersion oil 116 disposed between the substrate 102 and a top lens 118 of the objective 114.
A common means of achieving objective-based TIRF microscopy is to focus the illumination light 100 travelling along an optical axis 115 of the microscope to a focal point near the outer edge of the objective 114 and at a back focal plane 120 of the microscope objective 114, as illustrated in FIG. 1. It should be noted that, although the back focal plane 120 is illustrated in a location that is external to the objective 114, it may alternatively be located within the objective 114. The objective 114 has a high numerical aperture (NA) in order to allow the illumination light 100 to be transmitted near the outer edges of the lenses of the objective 114 and directed into the substrate 102 with an angle of incidence 108 that supports total internal reflection. The substrate 102 and the immersion oil 116 may have nearly the same refractive index n1, for example, approximately 1.52, and the specimen 106 may be in an aqueous medium with a refractive index n2 of approximately 1.33 to 1.40, for example, which supports total internal reflection within the substrate 102. The NA of the objective 114 is higher than the refractive index n2 of the specimen 106. The illumination light 100 strikes the substrate/specimen interface 104 with an angle of incidence 108 greater than the critical angle and is reflected back into the substrate 102 at the interface 104. The illumination light 100 creates in the specimen 106 an evanescent electromagnetic field adjacent to the interface 104.
The radial distance, for example the distance 122 in FIG. 1, of the point of light from the optical axis 115 of the objective 114 determines the angle that the light will have when leaving the objective 114. This, in turn, affects the angle of incidence 108 at the substrate/sample interface 104. Light focused further from the optical axis 115 will have a larger angle of incidence 108. By adjusting the position that the light focuses onto the objective back focal plane 120, the angle of incidence 108 can be adjusted to be near or slightly larger than the critical angle. The degree to which the angle of incidence 108 is greater than the critical angle will determine the depth of the evanescent wave and thus the imaging depth. These instruments, which use oil-immersion objectives with a high numerical aperture, are increasing in popularity today.
TIRF microscopy is an established microscopy technique with a number of implementations. FIG. 2 illustrates a possible simple TIRF implementation with a single mode fiber light delivery subsystem.
Illumination light from a single mode fiber 200 is collimated using a lens 202, and then directed via a lens 204 to a dichroic mirror 206. Illumination light incident on the dichroic mirror 206 is focused onto a back aperture 208 of an objective 210 at a desired radial distance R 212 from the optical axis 213 of the objective 210. The radial distance R 212 is adjustable by laterally moving optical elements such as the single mode fiber 200 or the lens 202 or the lens 204, where the lateral direction is denoted by an arrow 224 in FIG. 2.
The objective 210 directs the illumination light, via a hemispherical lens 222, through a substrate 214 and into a sample 216 to be imaged. The illumination light may strike the interface between the substrate 214 and the sample 216 with angles of incidence that are greater than the critical angle, such that total internal reflection is achieved.
Fluorescent light emitted from the sample 216 near the substrate/sample interface may be captured by the hemispherical lens 222 of the microscope objective 210 at the operating numerical aperture NA of the microscope objective 210. The collected fluorescent light further passes through the dichroic mirror 206 and is focused by a tube lens 218 onto an image plane which coincides with an image sensor of an imaging device 220.
Varying an incidence angle of the illumination light or a depth along which observation should be carried out is usually accomplished in the objective-based TIRF microscopy instrument by varying the radial distance of the focused light spot of the illumination light at the back focal plane of the microscope objective. The lateral displacement can be implemented through any of a plurality of technically simple means. For example, in such a microscope, a pick-off member, which reflects the light from a light source to a sample, may be placed in the back focal plane. The pick-off member may be in the form of a small mirror, as described in JP9159922A. Alternatively, the pick-off member may be in the form of a right angle prism, as described in U.S. Pat. No. 6,987,609. A displacement of the pick-off member in the radial direction away from the optical axis of the objective leads directly to a corresponding change in the angle of incidence of the illumination light and the penetration depth of the TIRF imaging.
In another example, radial beam displacement may be achieved using deflection means such as a steering mirror (as described in JP2002031762) or an acousto-optical modulator (as described in US Patent Application Publication No. 20030058530), in combination with a focusing lens. The radial beam adjustment may be done by a lateral movement of the tip of a light delivering optical fiber, as described in U.S. Pat. No. 6,924,490, or by lateral movement of a focusing lens, as described in U.S. Pat. No. 6,992,820. The TIRF microscope described in U.S. Pat. No. 7,224,524 comprises an optical device in the form of a wedge plate which is disposed on the optical path of the optical illumination system and de-centers an optical axis of the light beam.